Astrocytes retain the potential to divide throughout development, and during brain disease and repair. The central hypothesis of this proposal is that for astrocytes dividing during maturation or after months of quiescence, the G1/S transition of the cell cycle is regulated by the covalent modification of proteins by nonsterol isoprenoid lipids derived from mevalonate (i.e.,isoprenylation). Isoprenylation occurs on several polypeptides involved potentially in cell division, including ras proteins and related GTPases, and the nuclear lamins. To evaluate this hypothesis, cultured rat astrocytes at three presumably different maturational stages will be synchronized by serum deprivation: 1. primary newborn brain cultures within days of explantation, when the constituent astroglia are known to reflect the postnatal surge in glial proliferation; 2. secondary astrocytes obtained after 2-3 mos. in primary culture, which in preliminary experiments were found to recapitulate certain features of gliosis; 3. primary astroglia obtained from gelfoam implants into traumatized, gliotic areas of adult brain. Cells progressing through G1 will be treated with mevinolin. In each culture paradigm, this inhibitor of mevalonate biosynthesis causes cell cycle arrest which is reversed by brief exposure to mevalonate in late G1. Reversal of arrest is associated with relatively intense isoprenylation of peptides having molecular masses of 20-30 kD. Cultured astrocytes were found to contain several small, non- ras GTPases in this range. To determine which proteins are isoprenylated in the specific context of commitment to the G1/S transition, cultures will be exposed at different cell cycle times to [3H] mevalonate after mevinolin treatment. Combined [3H]mevalonate and [35S]methionine labeling and immunoprecipitation with specific antibodies against p21(ras) proteins, non-ras GTPases and nuclear lamins will be used to confirm isoprenylation, and to specify the appearance during the cell cycle of substrate proteins in relation to their isoprenylation. One and 2 dimensional peptide mapping and where necessary, sequencing of specific proteins, will be used to further establish protein identity at these times. The relationship between appearance and isoprenylation of specific proteins will be studied in the different astrocyte culture systems. These experiments will provide new information regarding initiation of cell cycling in early developing cultured astrocytes, with comparison to astrocytes that resemble glial cells proliferating in response to pathologic conditions in mature brain.
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